Total protein was extracted by lysing cells with RIPA lysate I (C5000050010; Sangon Biotech, Shanghai, China). Protein concentration was measured using a BCA Protein Assay Kit (C5030210500; Sangon Biotech, China). Equal amounts of protein lysate were boiled for 5 minutes with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (P1040; Solarbio, Beijing, China) and separated by SDS-PAGE Preparation kit (C631100; Sangon Biotech, China) and transferred to polyvinylidene fluoride membrane (Millipore ImmobilonTM P, Billerica, MA, USA). After blocking in 5% skim milk dissolved in Trisbuffered saline (0.02 M Tris base, 0.14 M NaCl, pH 7.4) containing 0.1% Tween 20, the membrane was incubated with primary antibodies overnight at 4°C, and then with IRDye 800CW goat anti-rabbit immunoglobulin G (Li-COR Biotechnology, Lincoln, NE, USA) for 1 hour at room temperature. The primary antibodies used in this study were UCP1 (ab10983; Abcam, USA), myf5 (ab139523; Abcam, USA), PPARγ (bs4590R; Bioss, China), CEBPα (GTX100674; Gene Tex, USA), β-actin (bs0061R; Bioss, China) and glyceraldehyde-3-phosphate dehydrogenase (HC301; TransGen, Beijing, China). The blots were imaged by the Odyssey CLx Imaging System (Li-COR Biotechnology, USA) and the protein density analysis was performed using Image J (v1.45) software.
Sds page preparation kit
Manufactured by Sangon
Sourced in China
The SDS-PAGE Preparation kit is a laboratory equipment used for the analysis of protein samples. It provides the necessary components for the separation and visualization of proteins based on their molecular weight using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx imaging system, by LI COR (1 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ab10983, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit immunoglobulin g, by LI COR (1 mentions)
β actin bs 0061r, by Bioss Antibodies (1 mentions)
Gtx100674, by GeneTex (1 mentions)
C ebpα, by GeneTex (1 mentions)
Hc301, by Transgene (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Dna marker, by Sangon (1 mentions)
Protein markers, by Sangon (1 mentions)
Dna gel extraction kit, by Sangon (1 mentions)
Bamhi, by Takara Bio (1 mentions)
T5 super pcr mix, by Tsingke (1 mentions)
Rapid yeast genomic dna isolation kit, by Sangon (1 mentions)
Ts gelred nucleic acid dye, by Tsingke (1 mentions)
Amicon ultra 15 centrifugal filter device, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
7 protocols using sds page preparation kit
1
Western Blot Analysis of Protein Expression
2
Yeast Genomic DNA Extraction and Manipulation
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DNA restriction enzymes (BamHI, NotI, and SacI) were purchased from Takara Biomedical Technology Co., Ltd (Beijing, China). The Rapid Yeast Genomic DNA Isolation Kit, Plasmid DNA Mini-preps Kit, SDS-PAGE Preparation Kit, DNA Markers, Protein Markers, DNA Gel Extraction Kit, and the primers for α-factor, 5′-AOX, and 3′-AOX were supplied by Sangon Biotech Co., Ltd (Shanghai, China). TS GelRed Nucleic Acid Dye and T5 Super PCR Mix were obtained from Beijing Tsingke Biotech Co., Ltd (Beijing, China). Hemin (CAS: 16009-13-5, purity: 98%) and biliverdin hydrochloride (CAS: 55482-27-4, purity: 97%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Geneticin (G418) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). All other reagents were commercially available in analytical and biological grades.
3
Cloning, Expression, and Purification of Proteins
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The plasmid pET30a(+)-6× His-SC, pET28a(+)-6× His-ST-GFP, and the competent Escherichia coli (E. coli) strain BL21(DE3) were obtained from our laboratory. Na2HPO4·12H2O, NaH2PO4·2H2O, CaCl2, NaCl, HCl, NaOH, and ethanol were purchased from Sinopharm (Beijing, China). EDC, Sulfo-NHS, kanamycin sulfate, agar, isopropyl-β-D-thiogalactopyranoside (IPTG), imidazole, glycine, 2-(N-morpholino)ethanesulfonic acid (MES), BSA, SDS-PAGE preparation kit, Bradford reagent, and carboxyl-SMBs (300 nm in diameter) were purchased from Sangon Biotechnology (Shanghai, China). A 0.1 M phosphate-buffered saline solution (PBS, pH 7.4) was prepared with 29 mg/mL Na2HPO4·12H2O, 2.965 mg/mL NaH2PO4·2H2O, and 8.766 mg/mL NaCl in distilled water, and the pH was adjusted to 7.4 by 1.0 M HCl. PBS (0.02 M) was prepared with 5.8 mg/mL Na2HPO4·12H2O, 0.593 mg/mL NaH2PO4·2H2O, and 29.22 mg/mL NaCl in distilled water, and the pH was adjusted to 7.4 by 1.0 M HCl. HisTrap™ HP 5 mL columns were obtained from GE Healthcare (Pittsburgh, PA, USA). Amicon® Ultra-15 centrifugal filter devices were purchased from Merck Millipore (Darmstadt, Germany).
4
Protein Molecular Weight Profiling
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SDS-PAGE was carried out to estimate the molecular weight profiles of protein and hydrolysates by using the SDS-PAGE Preparation kit (Sangon Biotech, Shanghai, China). Protein sample and loading buffer (Beyotime, Shanghai, China) were mixed in a 4 : 1 ratio and incubated in boiling water bath for 10 min. Aliquots of 10 μL were loaded into the gels (5% stacking gel and 15.5% separating gel). The electrophoresis was performed in a Mini-PROTEAN Tetra Cell system (Bio-Rad, USA).
5
SDS-PAGE Analysis of Corona Complexes
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SDS-PAGE was carried out according to standard procedures48 (link). Corona complexes were separated by 12% SDS-PAGE using the SDS-PAGE Preparation Kit (Sangon Biotech Co., Ltd, Shanghai, China). Then proteins were visualized by staining with Coomassie brillian blue R-250. All experiments were conducted at least twice to ensure the reproducibility of the results.
6
Immunoblotting Protocol for Protein Detection
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After treatment with 5× SDS-PAGE loading buffer (Beyotime Biotechnology; Cat# P0015) at 98 °C for 10 min, samples were resolved on 8%, 10% or 12% SDS polyacrylamide gels from the SDS-PAGE preparation kit (Sangon Biotech, Shanghai, China; Cat# C631100) and electroblotted onto polyvinylidene fluoride western blot membranes (Merck Millipore KGaA, Darmstadt, Germany; Cat# 03010040001). Membranes were incubated in BeyoECL Moon detection reagent (Beyotime Biotechnology; Cat# P0018FS) and visualized using the Amersham Imager 600 (AI600 RGB; GE Healthcare, Uppsala, Sweden) after application of primary rabbit anti-Flag antibody (Sigma-Aldrich; Cat# F7425), rabbit anti-HA antibody (Cell Signaling Technology, Danvers, MA, USA; Cat# 3724; lot# 10), rabbit anti-eIF4G antibody (Cell Signaling Technology; Cat# 2498; lot# 4), rabbit anti-eIF4E antibody (Cell Signaling Technology; Cat# 2067; lot# 8), rabbit anti-GAPDH (6C5) antibody (Santa Cruz Biotechnology; Cat# sc-32233; lot# L2019) or rabbit anti-Bax antibody (Cell Signaling Technology; Cat# 14796; lot# 800) and secondary HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology; Cat# A0208; lot# 110219200406).
7
Optimized SDS-PAGE Gel Staining Protocol
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The SDS-PAGE Preparation kit (Sangon Company, Shanghai, China) was used for polyacrylamide gel electrophoresis. The electrophoresis buffer was prepared by diluting 10 ×TBE (100 ml) in 900 ml of distilled water. The electrophoresis program began with 40 min of pre-electrophoresis followed by 70 min of electrophoresis after sample loading. The sample loading volume was 10 µl. The gel silver staining method was modified from Borzooeian et al. (2018) (link). The method comprises of settling with 100 ml/L acetic acid (CH3COOH) for 20 min, washing with ddH2O for 1 min and silver staining with 1 g/L of silver nitrate solution (AgNO3) for 20 min. The sample was then washed with ddH2O for 10 s, developed using 16 g/L sodium hydroxide (NaOH), 8 ml/L formaldehyde (37%) solution for 5 min, a wash with ddH2O for 10 s and a quenching reaction via the use of 7.5 g/L sodium carbonate (Na2CO3) for 5 min. The AFLP molecular marker bands were counted regardless of their presence or absence at the same position, with their presence marked as ‘1’ and absence marked as ‘0’. A binary data ‘0-1’ matrix was then established.
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